The invention is directed to methods of separating and isolating protein from biological samples comprising cells. More specifically, the invention is directed to methods of separating and extracting protein from different subcellular compartments of cells in a biological sample. The invention is also directed to a kit for isolating and extracting protein from different subcellular compartments of cells in a biological sample.
Protein is a basic component of cells and the subject of a vast area of research. Location of a protein within a cell is related to the function of the protein. For example, a protein involved in intracellular signal transduction would be expected to be found in the cytoplasmic compartment of a cell. A transcription factor protein would be found in the nuclear compartment of a cell, while a receptor would be found in the cell membrane. Therefore, isolation of a protein from a specific compartment of a cell (e.g., the cytoplasmic compartment, the nuclear compartment or the cell membrane compartment) is useful for studying the protein""s function. Determination of cellular compartmental localization is also a standard technique in the study of Functional Genomics.
Many assays are used in the study of protein function. These include Western Blot, immunoprecipitation, receptor binding assays, protein-DNA and protein-mRNA binding assays, transcription assays, signal transduction assays, enzyme activity assays, phosphorylation and dephosphorylation analysis, etc. These assays generally require that the structure and activity of a protein of interest be maintained. The methods used to isolate and extract protein from its source should be compatible with the procedures to which the protein will subsequently be subjected. Ideally, the methods of isolation and extraction used should maintain the primary (sequence), secondary (folding) and tertiary (subunits) structure of the protein. This is best achieved with a minimum of manipulation of the protein during the isolation and extraction process.
In addition to preserving structure and activity of protein, several other parameters are also important in the choice of a protein isolation and extraction process. These include recovery rate, quickness and ease of use, and scalability. Of further importance is maintenance of the ratio of protein extracted from different subcellular compartments of a sample for gene expression analysis. The current methods of isolation and extraction of protein from a biological sample include density gradient centrifugation, ultra-centrifugation, concentration, dialysis, chromatography, precipitation, electrophoresis and selective banding. Unfortunately, each of these methods has one or more shortcomings with regard to the parameters listed above. Effective methods of extracting nuclear protein are available, but they generally exclude isolation and extraction of proteins from the cytoplasmic and membrane compartments of a cell.
Due to the broad interest in protein isolated from specific subcellular compartments and the importance of the various considerations described above for the means of such protein isolation and extraction, there is a need for a simple and effective method of isolating and extracting protein from different subcellular compartments of biological samples. A method that maintains protein structure and function, as well as the natural ratio of proteins, from different subcellular compartments can be applied to many different types of investigation of protein function. Accordingly, the present invention provides quick, simple and widely applicable methods for the isolation and extraction of protein from different subcellular compartments of biological samples.
The present invention provides methods of separating and isolating protein from at least three subcellular compartments of a single biological sample. The invention further applies the characterization of the subcellular distribution of proteins for fingerprinting and identifying different types of biological samples.
In one aspect, the invention provides a method of separating and isolating protein from at least three different subcellular compartments of a biological sample. In a preferred embodiment, the sample is homogenized and incubated in a cytoplasmic protein extraction solution, then centrifuged, and the supernatant containing the cytoplasmic protein is extracted. The pellet is then resuspended in a nuclear protein extraction solution, incubated, centrifuged, and the nuclear protein is extracted. The pellet is then resuspended, incubated in a membrane protein extraction solution, incubated, centrifuged, and the membrane protein is extracted. Preferably, the cytoplasmic protein extraction solution is a low osmotic solution, the nuclear protein extraction solution is a high osmotic solution, and the membrane protein extraction solution is a low osmotic solution containing a surfactant. In a preferred embodiment, the nuclear protein extraction solution contains at least one DNase. Preferably, each of the solutions contain protease inhibitors. Preferably, all of the incubation and centrifugation steps are performed in the same container, and preferably sequentially so as to be considered one procedure. In a preferred embodiment, the amounts of protein in each protein-containing supernatant is measured, whereby the total protein per weight of sample and the relative distribution of protein in each subcellular compartment is determined. The biological sample may be of organisms, tissue or cells, as long as the sample contains cells.
In another aspect of the invention, provided herein are methods of determining the subcellular compartmentalization of one or more proteins of interest in a biological sample. The protein from at least three subcellular compartments is separated and isolated, as described above. The presence of the protein(s) of interest in the protein isolated from one or more of the subcellular compartments is then determined. The determination of the presence of the protein(s) of interest may be by any of the many methods known in the art for identifying a specific protein.
The invention also provides methods of fingerprinting a biological sample. Protein from at least three subcellular compartments of the sample is separated and isolated, as described above. The pattern of protein present in the protein isolated from each subcellular compartment is then determined and the patterns are used to fingerprint the sample.
In a further aspect of the invention, methods of identifying the type of a biological sample are provided. Protein from subcellular compartments of the sample are separated and isolated as above, as is the pattern of protein present in the protein isolated from each of the subcellular compartments. Alternatively, these patterns are compared to patterns of protein from the same subcellular compartments in biological samples of known type.
Also provided are methods of identifying cancerous tissue or cells. Protein from subcellular compartments of the sample are separated and isolated as above, as is the pattern of protein present in the protein isolated from each of the subcellular compartments. Alternatively, these patterns are compared to patterns of protein from the same subcellular compartments in known normal and/or known cancerous tissue of the same type as the sample. The cancerous tissue or cells may be metastatic or of a tumor.
In a further aspect of the invention, provided herein are methods of identifying the developmental stage of a tissue sample. Protein from subcellular compartments of the sample are separated and isolated as above, as is the pattern of protein present in the protein isolated from each of the subcellular compartments. Alternatively, these patterns are compared to patterns of protein from the same subcellular compartments in tissue of the same type and of known developmental stage.
The invention also provides a kit for separating and isolating subcellularly compartmentalized proteins from a biological sample. The kit contains a cytoplasmic extraction solution, a nuclear protein extraction solution and a membrane protein extraction solution. The kit may also contain means for separating and isolating protein from each of the cytoplasmic compartment, the nuclear compartment and the membrane compartment of the biological sample.